Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is a key enzyme in the de novo synthesis of purines. IMPDH catalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP), resulting in the production of NADH and XMP. IMPDH catalyzed oxidation of IMP to XMP is the rate-limiting step in the synthesis of guanine nucleotides.
IMPDH exists as a homotetramer (i.e., the enzyme has four subunits each comprising an IMPDH polypeptide). In many species, two isoforms of IMPDH have been described and are designated type I and type II IMPDH. Human type I and type II IMPDH have been identified and sequenced. Human IMPDH types I and II are both 514 amino acids in length and they share 84% sequence identity. The nucleotide and amino acid sequence of human type I IMPDH are disclosed in Natsumeda, Y., et al., J. Biol. Chem. 265:5292-5295 (1990), and the nucleotide and amino acid sequence of human type II IMPDH are disclosed in Natsumeda, Y., et al., J. Biol. Chem. 265:5292-5295 (1990), Collart, F. R. and Hubermann, E., J. Biol. Chem. 263:15769-15772 (1988), and U.S. Pat. No. 5,665,583. The subunits of human IMPDH types I and II that make up the IMPDH homotetramer each have a subunit molecular weight of 56 kDa. Human type II IMPDH has a catalytic core domain (amino acids 1-109 and 245-514) and a subdomain (amino acids 110-244) with unknown function.
IMPDH is a target for antitumor (e.g., antileukemic) therapy and immunosuppressive chemotherapy. IMPDH is upregulated in neoplastic and differentiating cells. Furthermore, proliferating B and T lymphocytes depend on the de novo pathway, rather than the salvage pathway, for synthesis of guanine nucleotides with inhibition of guanine nucleotide synthesis resulting in inhibition of DNA synthesis. Thus, IMPDH is an important enzyme for B and T cell proliferation, and inhibition of IMPDH activity inhibits both B and T cell proliferation making IMPDH an important target for immunosuppressive chemotherapy.
Mycophenolic acid (MPA) is an uncompetitive inhibitor of human types I and II IMPDH and MPA binds IMPDH after NADH is released, but before XMP is produced. MPA is the active metabolite in vivo of the ester prodrug mycophenolate mofetil (CellCept; MMF). MMF is an immunosuppressant that blocks B and T cell proliferation and MMF has been approved for the treatment of kidney and heart transplant rejection. MMF has also been used clinically to treat cancer and viral infections, and has been used as an anti-vascular hyperproliferative agent, an antipsoriatric agent, an antibacterial agent, an antifungal agent, and has been used for the treatment of autoimmune diseases.
MMF is hydrolyzed to MPA in vivo, and, accordingly, the monitoring of MPA levels in vivo allows for monitoring of MMF dosages. The measurement of MPA levels in patients treated with MMF is of clinical significance because the monitoring of MPA levels improves therapeutic efficacy, e.g., optimal MMF levels necessary for adequate immunosuppression can be determined, and minimizes the adverse side effects of the drug. Isolated, recombinant IMPDH has been used in assays to measure MPA levels in patients treated with MMF, such as the assays described in U.S. Pat. Nos. 6,107,052 and 6,524,808.
The ability to produce and to isolate large amounts of stable, recombinant IMPDH (e.g., IMPDH that aggregates minimally) is important for use in assays for monitoring MPA levels in patients treated with MMF, or for use in assays to monitor the levels in patient samples of any other therapeutically useful IMPDH inhibitor. Other inhibitors of IMPDH are described in Anderson, J. H. et al., J. Biol. Chem. 243:4762-4768 (1968) and in U.S. Pat. Nos. 5,380,879, 5,444,072, and 5,807,876 and in PCT publications WO 94/01105 and WO 94/12184.
The ability to produce and to isolate large amounts of stable, recombinant IMPDH is also important for other clinical and research applications, such as for the identification and design of new IMPDH inhibitors useful for cancer and immunosuppressive therapies, and for determining the sensitivity of IMPDH to those inhibitors.